Role of bid in liver injury following BDL and CCl4-induced liver damage [Elektronische Ressource] / Padmavathi Devi Nalapareddy

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Role of Bid in Liver injury following BDL and CCl4-induced liver damage der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover Zur Erlangung des Grades Doktorin der Naturwissenschaften Dr.rer.nat. Genehmigte Dissertation Von M.Sc. Padmavathi Devi Nalapareddy Geboren am 05.06.1979 in Tirupati, India. 1 The following study has been carried out under the supervision of Dr. med. Arndt Vogel, Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany between October 2006 and October 2009. Declaration: Here with I declare that the study has been done by my own under the guidance of Dr. med. Arndt Vogel and all the information provided is novel and true and has not been submitted to any other institute or University to obtain any other degree. Padmavathi devi Nalapareddy Primary Referee :Prof. Dr. S. Kubicka, Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl-Neuberg-Str. 1,30625 Hannover Co-referee :Prof. Dr.rer.nat. Ralf Hass, AG Biochemie und Tumorbiologie. Klinik für Frauenheilkunde und Geburtshilfe Medizinische Hochschule Hannover Co-referee-2 :PD. Dr.

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Role of Bid in Liver injury following BDL and
CCl4-induced liver damage











der Naturwissenschaftlichen Fakultät der
Gottfried Wilhelm Leibniz Universität Hannover
Zur Erlangung des Grades Doktorin der Naturwissenschaften



Dr.rer.nat.



Genehmigte Dissertation



Von



M.Sc. Padmavathi Devi Nalapareddy



Geboren am 05.06.1979 in Tirupati, India.








1 The following study has been carried out under the supervision of Dr. med. Arndt Vogel,
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School,
Hannover, Germany between October 2006 and October 2009.






Declaration: Here with I declare that the study has been done by my own under the guidance
of Dr. med. Arndt Vogel and all the information provided is novel and true and has not been
submitted to any other institute or University to obtain any other degree.




Padmavathi devi Nalapareddy




Primary Referee :Prof. Dr. S. Kubicka,
Department of Gastroenterology, Hepatology and Endocrinology,
Hannover Medical School,
Carl-Neuberg-Str. 1,30625 Hannover


Co-referee :Prof. Dr.rer.nat. Ralf Hass, AG Biochemie und Tumorbiologie.
Klinik für Frauenheilkunde und Geburtshilfe
Medizinische Hochschule Hannover


Co-referee-2 :PD. Dr. Jochen Heß
Signal Transduction And Growth Control
German Cancer Research Center - DKFZ
Im Neuenheimer Feld 280
69120 Heidelberg
Germany
Tel: -49 -6221-42 4501
Fax: -49-6221-42 4554
Email: j.hess@dkfz.de


Tag der promotion: 11 February 2010


2





Acknowledgements

I am very grateful to my supervisor Dr. med. Arndt Vogel for giving me the opportunity,
encouragement and support though out my doctorial work to pursue my research and also I
am grateful to Prof. Dr. M. P. Manns for giving me opportunity to pursue my research in
Hannover Medical School. I would like to Thank Prof. Dr. S. Kubicka, Department of
Gastroenterology, Hepatology & Endocrinology for the help to pursue my doctorial thesis.

I would like to thank my colleagues Deepika, Jessica, Jutta, Laura, Silke and Sven for
valuable discussions and co-operation through out my research work.

I would like to thank my husband N. Kodandaramireddy for valuable discussions and co-
operation through out my research work.























3




Contents

Title page 1
Declaration 2
Acknowledgements 3
Contents 4
Abstract 7
1. Introduction 10
1.1. Apoptosis 10
1.1.1. Death receptor pathway or extrinsic pathway 10
1.1.2. Mitochondrial or intrinsic pathway 11
1.2. The Bcl-2 family proteins as important apoptosis regulators 11
1.3. Bid Protein and its role in liver 11
1.4. Molecular mechanisms of Bid in mitochondrial pathway 12
1.5. Role of Bid in Fas induced apoptosis 13
1.6. Role of Bid in FasL/Mega Fas induced apoptosis 14
1.7. Role of Bid in Tnf-R mediated apoptosis 14
1.8. Role of apoptosis/Bid in liver disease 15
1.8.1. Cholestasis/ BDL 15
1.8.2. CCl4 model 16
1.9. Summary and focus of the current study 17
2. Materials and Methods 18
2.1. Mice 18
2.2. Genotyping 18
2.2.1. DNA Extraction from mouse tails 18
2.2.2. Genotyping for Bid mice 18
2.2.3. Genotyping for c-Jun mice 18
2.3. Serum measurements 20
2.4. Histology 20
2.4.1. Haematoxylin and Eosin (H&E) staining 20
2.4.2. Sirius red staining 20
2.4.3. Masson’s trichome staining 21
4

2.5. Immunohistochemistry 21
2.5.1. TUNEL assay as apoptosis marker 21
2.5.2. Cleaved caspase 3 staining as a marker of apoptosis 21
2.5.3. BrdU staining as proliferation marker 22
2.5.4. Ki 67 staining as proliferation marker 22
2.5.5. Alpha-SMA staining as a fibrosis marker 23
2.5.6. Naphthol AS-D chloroacetate esterase staining for neutrophills 23
2.6.1. CD11b staining for neutrophill 24
2.5.8. CD68 staining for Kupffer cells 24
2.6. Western blot 24
2.6.1. Protein preparation 24
2.6.2. Protein separation through SDS-PAGE and western blotting 24
2.7. Real-time RT PCR for mRNA level expressions 25
2.7.1. Isolation of total RNA using Trizol 25
2.7.2. cDNA synthesis and real time RT PCR 25
2.8. Caspase-3 activity assay 26
2.9. Measurement of Hepatic Hydroxyproline Content 26
3.0. Cell culture method 27
3.1. Isolation of primary hepatocytes 27
3.2. Treatment of hepatocytes 27
3.3. Apoptosis assay on hepatocytes- TUNEL staining 28
4.0. Antibodies 28
4.1. Cell culture reagents 29
4.2. Chemicals 29
4.3. Enzymes, DNA ladder and protein ladder 30
4.4. Laboratory equipment 30
4.5. Kits 31
4.6. Statistical analysis 31

5.0. Results 31
5.0. The BH3-only protein bid does not mediate death-receptor-induced liver injury in
obstructive cholestasis.
5.1. Loss of Bid does not affect liver injury and overall survival of mice following BDL 31
5.2. Loss of B affect apoptosis of hepatocytes following BDL mice 33
5

5.3. Loss of Bid does not affect proliferation of hepatocyte following BDL 35
5.4. Loss of B attenuate fibrogenesis following BDL 36
5.5. Loss of Bid does not affect the inflammatory response following BDL 38
-/-5.6. Bid and lpr mice display similar activation of stress kinase pathways
following BDL 39
6.0. Role of Bid in liver injury following CCl4 41
6.1. Loss of Bid attenuates liver damage following acute dose of CCl4 injection 41
6.2. Loss of Bid doesn’t affect apoptosis following acute dose of CCl4 injection 42
6.3. Loss of Bid in proliferation following acute CCl4 injection 43
-/-6.4. Loss of Bid attenuates the immunological response in Bid mice following
acute CCl4 damage 45
6.5. Loss of Bid accelerates liver injury following chronic liver damage 47
6.6. Loss of Bid in Fibrosis following chronic dose of CCl4 48
6.7. Loss of Bid in proliferation after chronic dosage of CCl4 49
7.0. Discussion 50
Reference List 53
Curriculum Vitae 61






















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Abstract:

Death receptor-mediated hepatocyte apoptosis has been implicated in a variety of acute
and chronic liver diseases. The BH3-interacting domain death agonist Bid is a critical
mediator for apoptosis induced by activation of Fas and TNF-R1 death receptors in
hepatocytes. The aim of this study was therefore to elucidate the role of Bid in cholestatic and
toxic liver injury.
Accumulation of bile acids during obstructive cholestasis causes liver injury and fibrosis,
which is at least partly mediated by the death receptors Trail, Tnf-  and Fas. To analyse the
role of Bid in cholestatic liver injury, overall survival and various aspects of liver injury were
-/-analyzed in WT and Bid mice following bile duct ligation, a commonly used model to study
obstructive cholestasis in mice. Loss of Bid did not affect number of bile infarcts, serum AST
-/-values and animal survival. Importantly, Bid mice displayed the same pattern of TUNEL
positive hepatocytes as WT controls. Processing of procaspase-3 and -9 and caspase-3
enzyme activities however were not detectable in either group. In contrast to Fas-receptor
deficient lpr mice, hepatic fibrosis and the inflammatory response was not affected by loss of
Bid. Together, these data suggest that Bid does not play a role in cholestatic liver injury.
-/- To analyse the role of Bid in toxic liver injury, WT and Bid mice were challenged with
CCl4. Previously, it has been proposed that CCl4 does not only causes necrosis, but also
induces apoptosis in mouse livers, which might sustain inflammation and liver injury. Here
we show that loss of Bid ameliorates CCl4 induced liver injury even though CCl4 did not
induce significant hepatocyte apoptosis. Reduced liver injury after a single CCl4 injection
correlated with an attenuated inflammatory response, which might contribute to the observed
phenotype. Surprisingly, loss of Bid caused more severe liver injury in mice treated for 6
weeks with CCl4 suggesting that Bid is required for the adaptation of hepatocytes against
chronic liver injury induced by CCl4. Additional studies will be necessary to further dissect
the role of Bid in toxic CCl4-induced liver injury. Our data however indicate that Bid plays a
role in the liver beyond its role in mediating hepatocyte apoptosis.
Key words: Bid, BDL and CCl4



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Abstrakt:

Todesrezeptor-vermittelte Apoptose von Hepatozyten spielt eine wichtige Rolle in
einer Reihe von akuten und chronischen Lebererkrankungen. Bid (BH3-interacting
domain death agonist) ist ein kritischer Vermittler für Apoptose, die durch Aktivierung
von Fas und TNF-R1 Todesrezeptoren in Hepatozyten induziert wird. Ziel dieser Studie
war es deshalb die Rolle von Bid in cholestatischen und toxischen Leberschädigungen zu
ermitteln.
Die Akkumulation von Gallensäuren während obstruktiver Cholestase
verursacht eine Leberschädigung und -fibrose. Um die Rolle von Bid während einer
cholestatischen Leberschädigung zu analysieren, wurden das Überleben und
-/-verschiedene Aspekte der Leberschädigung in WT und Bid Mäusen nach
Gallengangligatur, ein etabliertes Modell zur Untersuchung der obstruktiven Cholestase
in Mäusen, analysiert. Der Verlust von Bid beeinflusste weder die Anzahl von
Galleninfarkten und die Höhe der AST Serumwerte noch das Überleben der Tiere.
-/-Interessanterweise fanden sich zudem in Lebern der Bid Mäuse die gleiche Anzahl
und Verteilung von TUNEL positiven Hepatozyten im Vergleich zu den WT Kontrollen.
Eine Prozessierung von Procaspase-3 und -9 und Caspase-3 Enzymaktivitäten war
jedoch in keiner der Gruppen detektierbar. Anders als in Fas-Rezeptor defizienten lpr
Mäusen wurden durch den Verlust von Bid weder die hepatische Fibrose noch die
inflammatorische Antwort beeinflusst. Zusammenfassend lässt sich vermuten, dass Bid
keine Rolle in der cholestatischen Leberschädigung spielt.
Um die Rolle von Bid in der toxischen Leberschädigung zu analysieren, wurden
WT und Bid-/- Mäuse mit CCl4 behandelt. Früheren Arbeiten lassen vermuten, dass
CCl4 nicht nur Leberzellnekrosen auslöst, sondern auch Apoptose von Introduction
Hepatozyten induziert, wodurch möglicherweise die Entzündungsreaktion und damit
die Leberschädigung verstärkt wird. Wir konnten in dieser Arbeit zeigen , dass der
Verlust von Bid zu einer Verbesserung des durch CCl4 induzierten Leberschadens
führt, obwohl CCl4 keine signifikante Apoptose in Hepatozyten auslöste. Die reduzierte
Leberschädigung nach einfacher CCl4-Injektion korrelierte mit einer verzögerten
Immunantwort, welche zu dem beobachteten Phänotyp beitragen könnte.
Überraschenderweise verursachte der Verlust von Bid einen stärkere Leberschädigung
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in Mäusen, die über sechs Wochen mit CCl4 behandelt wurden, was die Vermutung
nahelegt, dass Bid für die Adaption von Hepatozyten gegenüber CCl4-induzierter
chronischer Leberschädigung beiträgt. Zusätzliche Studie werden notwendig sein, um
die exakte Rolle von Bid im Rahmen der CCl4-induzierten Leberschädigung zu klären.
Insgesamt lassen unsere Ergebnisse aber vermuten, dass Bid in Hepatozyten nicht nur
Apoptose induziert, sondern noch weitere biologische Funktionen ausübt.
Schlagworter: Bid, BDL and CCl4



























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1. Introduction:
1.1 Apoptosis:
Apoptosis, programmed cell death (PCD), is a morphologically and biochemically
distinct form of cell death. Apoptosis can be characterized by membrane blebbing, chromatin
condensation and nuclear fragmentation. During apoptosis the intracellular organelles remain
intact in contrast to necrosis. Apoptosis may occur by two pathways, the death receptor or
extrinsic pathway and the mitochondrial or intrinsic pathway.

1.1.1 Death receptor pathway or extrinsic pathway:
The most extensively characterized death receptors are CD95 (Fas or Apo1) and
CD120a (TNF-R1) (Trauth et al., 1989); (Ito et al., 1991); (Ashkenazi and Dixit, 1998),.
Triggering of the CD95/Fas molecule either by agonistic antibodies or by the natural ligand
FasL induces trimerization of the receptor, and the trimerized cytoplasmic region then
transduces the signal into the cell by recruiting a molecule called FADD (Fas-associating
protein with death domain) or MORT1 (mediator of receptor- induced toxicity), which binds
to CD95 via interaction of the death domain at its COOH terminus (Chinnaiyan et al., 1996).
The NH terminal region of FADD is responsible for downstream signal transduction by 2
recruitment of a protein called FLICE (FADD Like Interleukin-1b Converting Enzyme) or
MACH (MORT1-Associated CED-3 Homologue), designated as caspase-8 (Boldin et al.,
1996).













The NH terminus of caspase-8 binds to FADD/MORT1, (while its COOH-terminal region is 2
10